Epac and phospholipase Cepsilon regulate Ca2+ release in the heart by activation of protein kinase Cepsilon and calcium-calmodulin kinase II.
Recently, we identified a novel signaling pathway involving Epac, Rap, and phospholipase C (PLC)epsilon that plays a critical role in maximal beta-adrenergic receptor (betaAR) stimulation of Ca2+-induced Ca2+ release (CICR) in cardiac myocytes. Here we demonstrate that PLCepsilon phosphatidylinositol 4,5-bisphosphate hydrolytic activity and PLCepsilon-stimulated Rap1 GEF activity are both required for PLCepsilon-mediated enhancement of sarcoplasmic reticulum Ca2+ release and that PLCepsilon significantly enhances Rap activation in response to betaAR stimulation in the heart. Downstream of PLCepsilon hydrolytic activity, pharmacological inhibition of PKC significantly inhibited both betaAR- and Epac-stimulated increases in CICR in PLCepsilon+/+ myocytes but had no effect in PLCepsilon-/- myocytes. betaAR and Epac activation caused membrane translocation of PKCepsilon in PLCepsilon+/+ but not PLCepsilon-/- myocytes and small interfering RNA-mediated PKCepsilon knockdown significantly inhibited both betaAR and Epac-mediated CICR enhancement. Further downstream, the Ca2+/calmodulin-dependent protein kinase II (CamKII) inhibitor, KN93, inhibited betaAR- and Epac-mediated CICR in PLCepsilon+/+ but not PLCepsilon-/- myocytes. Epac activation increased CamKII Thr286 phosphorylation and enhanced phosphorylation at CamKII phosphorylation sites on the ryanodine receptor (RyR2) (Ser2815) and phospholamban (Thr17) in a PKC-dependent manner. Perforated patch clamp experiments revealed that basal and betaAR-stimulated peak L-type current density are similar in PLCepsilon+/+ and PLCepsilon-/- myocytes suggesting that control of sarcoplasmic reticulum Ca2+ release, rather than Ca2+ influx through L-type Ca2+ channels, is the target of regulation of a novel signal transduction pathway involving sequential activation of Epac, PLCepsilon, PKCepsilon, and CamKII downstream of betaAR activation.
Oestreich
Emily A
EA
Department of Pharmacology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Malik
Sundeep
S
Goonasekera
Sanjeewa A
SA
Blaxall
Burns C
BC
Kelley
Grant G
GG
Dirksen
Robert T
RT
Smrcka
Alan V
AV
D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology; D019269:Phosphatidylinositol 4,5-Diphosphate; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D051744:Protein Kinase C-epsilon / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000502:physiology; D011943:Receptors, Adrenergic, beta / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D000099211:Phospholamban; D000096926:Benzenesulfonamides
D001596:Benzylamines; D002118:Calcium; D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D020662:Guanine Nucleotide Exchange Factors; D019269:Phosphatidylinositol 4,5-Diphosphate; D054801:Phosphoinositide Phospholipase C; D051744:Protein Kinase C-epsilon; D047428:Protein Kinase Inhibitors; D011943:Receptors, Adrenergic, beta; D019837:Ryanodine Receptor Calcium Release Channel; D013449:Sulfonamides; D000099211:Phospholamban; C482601:Epac protein, mouse; C072105:KN 93; D000096926:Benzenesulfonamides; C497256:Prkce protein, mouse; C423320:phospholipase C epsilon